Scientia Horticulturae 226 (2017) 353–360

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Research Paper

Foliar applications of a legume-derived protein hydrolysate elicit dose- MARK

dependent increases of growth, leaf mineral composition, yield and fruit
quality in two greenhouse tomato cultivars
⁎
Youssef Rouphaela, , Giuseppe Collab, Maria Giordanoa, Christophe El-Nakhela,
Marios C. Kyriacouc, Stefania De Pascalea
a
Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Italy
b
Department of Agricultural and Forestry Sciences, University of Tuscia, 01100 Viterbo, Italy
c
Department of Vegetable Crops, Agricultural Research Institute, 1516 Nicosia, Cyprus

A R T I C L E I N F O A B S T R A C T

Keywords: The use of natural plant biostimulants is proposed as a promising and innovative approach to ensure improved
Antioxidant activity and sustainable yields and product quality. A greenhouse experiment was performed to assess the yield per-
Lycopene formance, leaf net assimilation of CO2, mineral composition of leaves and fruits, and fruit physicochemical
Mineral composition quality attributes of two tomato cultivars (Akyra and Sir Elyan) in relation to biostimulant treatments (control or
Plant biostimulants
two different concentrations of the legume-derived protein hydrolysate Trainer®). Treated tomato plants were
Solanum lycopersicum L
Sustainable horticulture
sprayed every 10 days with a solution containing 2.5 and 5.0 ml L−1 of biostimulant. Akyra was found to be
richest in K, Ca, Mg, lipophilic and hydrophilic antioxidant activities (LAA and HAA), lycopene, total phenolic
and total ascorbic acid. Foliar applications of legume-derived protein hydrolysate at 5.0 ml L−1 increased
marketable yield of Akyra and Sir Elyan by modulating yield components differently depending on cultivars:
higher number of fruits in Akyra and increase of fruit mean weight in Sir Elyan. Improved yield performance
with biostimulant foliar applications at the highest rate was related to improved leaf nutritional status (higher K
and Mg) and higher net assimilation of CO2. The application of legume-derived protein hydrolysate at
5.0 ml L−1, and to a lesser degree at 2.5 ml L−1, elicited an increase in antioxidant activities, total soluble solids,
mineral composition (K and Mg) as well as bioactive molecules such as lycopene and ascorbic acid, thereby
increasing the nutritional and functional quality of the fruits. These findings can assist tomato growers in se-
lecting cultivars and application dose for protein hydrolysate to complement high crop productivity with optimal
fruit quality.

1. Introduction
In the coming years, food demand will rise steadily with con-
comitant increase in the global population. Thus, a fundamental chal-
lenge for agriculture is to produce sufficient food and, at the same time,
minimize collateral damage to the environment (Duhamel and
Vandenkoornhuyse, 2013). In other words, new farming practices
should be introduced in order to produce more food in a sustainable
way (i.e., by improving resource use efficiency). The use of natural
plant biostimulants has been proposed as a promising, safe and mean-
ingful approach to address the sustainability challenges facing horti-
culture and to ensure high yield and quality of horticultural commod-
ities (Colla and Rouphael, 2015; du Jardin, 2015). As defined by the
Association of American Plant Food Control Officials (www.aapfco.org)
plant biostimulants are ‘any substance or compound other than primary
(i.e., N, P, K) secondary (i.e., Ca, Mg, S) and microelement plant nutrients
(i.e., Fe, Mn, Zn, Cu), that can be demonstrated by scientific research to be
beneficial to one or more plant species when applied exogenously’.
According to Colla et al. (2015) protein hydrolysates (PHs) re-
present a well-known category of plant biostimulants (PBs) defined as
‘mixtures of amino acids, polypeptides and oligopeptides that are manu-
factured from animal or plant protein sources using partial hydrolysis’
(Schaafsma, 2009). They are available on the market as liquid products,
soluble powder or in granular form, and can be applied as seed treat-
ment, foliar spray and soil drench (Colla et al., 2015). PHs coming from
vegetal source of proteins are gaining interest worldwide because of
their superior agronomic value compared to animal-derived PHs
(Cerdán et al., 2009) and the lack of restrictions in the use of plant-

⁎
Corresponding author.
E-mail address: youssef.rouphael@unina.it (Y. Rouphael).

http://dx.doi.org/10.1016/j.scienta.2017.09.007
Received 1 August 2017; Received in revised form 1 September 2017; Accepted 8 September 2017
0304-4238/ © 2017 Elsevier B.V. All rights reserved.
Y. Rouphael et al.
derived PHs in organic farming.
Recent studies reported that foliar and/or root applications of PHs
are capable of eliciting an array of physiological processes in crops that
stimulate growth (Colla et al., 2014), enhance yield and product quality
(Colla et al., 2017; Ertani et al., 2014), improve tolerance to a wide
range of abiotic stresses, including drought (Feitosa de Vasconcelos
et al., 2009), salinity (Ertani et al., 2013; Lucini et al., 2015), thermal
(Botta, 2013) and nutrient stress (Colla et al., 2013) as well as adverse
soil pH (Rouphael et al., 2017a). Direct effects of PHs on plants include
fostering the activity of key enzymes (i.e., asparate aminotransferase,
glutamate synthase, glutamine synthetase, nitrate reductase and nitrite
reductase) involved in N reduction and assimilation as well as C me-
tabolism (Colla et al., 2015; du Jardin, 2015; Schiavon et al., 2008).
Moreover, PHs application could also elicit auxin- and gibberellin-like
activities due to the presence of bioactive peptides, thus boosting crop
performance (Colla et al., 2014; Ertani et al., 2009; Matsumiya and
Kubo, 2011). In addition to direct effects, the application of PHs has
been shown to modify root growth and architecture (i.e. indirect ef-
fects), facilitating the macro- and microelement uptake as a con-
sequence of the increased root surface area (density, length and number
of lateral roots; Colla et al., 2015; du Jardin, 2015; Ertani et al., 2009).
However, except from a few studies, the potential benefits from the
application of plant-derived PHs on vegetable crops were mainly in-
vestigated at tissue level (i.e. bioassays) and at seedling stage under
soilless and substrate culture (Colla et al., 2013, 2014; Lucini et al.,
2015; Matsumiya and Kubo, 2011). Limited information is available
concerning the effect of foliar applications of plant-derived PHs on
agronomical, physiological and fruit quality responses of an important
vegetable crop such as fresh-market tomato grown under soil condi-
tions. Moreover, the biostimulant action of PHs can vary depending on
species and/or cultivars, growing seasons, mode and dose of applica-
tions (Colla et al., 2015). Thus, understanding how the dose of appli-
cations and their interaction with cultivars can modulate crop pro-
ductivity and physico-chemical attributes of greenhouse tomato under
soil culture is an urgent need among vegetable farmers, extension
specialists and researchers for the continued success of the use of nat-
ural biostimulants.
Accordingly, the objectives of the current study were: i) to examine
the response of two greenhouse tomato cultivars to a legume-derived
PH, delivered by foliar application at three concentrations, in terms of
yield performance characteristics, leaf net CO2 assimilation rate, mi-
neral composition and fruit physicochemical composition, and ii) to
assess the associations between these nutritive traits.
2. Materials and methods
2.1. Experimental conditions and tomato cultivars
The experiment was carried out in the summer-autumn 2016, in an
unheated greenhouse covered by ethyl vinyl acetate 0.2 mm plastic
film, situated at Somma Vesuviana, Naples province, South Italy (lat.
40° 53′ 14” N, long. 14° 28′ 40” E; altitude 155 m above sea level). The
mean air temperature and relative humidity inside the greenhouse were
22 °C and 65%, respectively. The soil was sandy (85% sand, 7% silt, 8%
clay), with a bulk density of 1.1 g cm−3, pH of 7.5, electrical con-
ductivity of 1.2 dS m−1, organic matter of 0.8% (w/w), total N at
0.03%, available P at 16 mg kg−1 and exchangeable K at 35 mg kg−1.
Two tomato (Solanum lycopersicum L.) F1 hybrids were tested in the
current greenhouse experiment, cvs. Sir Elyan (Vilmorin INC, Tucson,
USA) and Akyra (Syngenta, Milan, Italy). The Sir Elyan cultivar is
characterized by a medium-size plum-shape fruit (70–100 g), good
potential for fruit setting and production and low sensitiveness to
blossom end rot, whereas the mini plum Akyra cultivar has an average
fruit weight between 30 and 40 g (small size) with 15–20 fruits per
truss, and characterized by its high resistance against cracking and its
long shelf life. Both cultivars are harvested at full ripening stage and are
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Scientia Horticulturae 226 (2017) 353–360
widely cultivated under protected cultivation around the world. At the
two true-leaf stage tomato seedlings were transplanted on 3 August
2016. Plant rows were 0.9 m apart, and the space between plants within
a row was 0.3 m. The distance between the centers of double rows was
2.22 m, resulting in a plant density of 3.0 plants m−2, as used com-
mercially for fresh tomato under greenhouse conditions.
2.2. Experimental design, protein hydrolysate application and crop
management
Six treatments derived by the factorial combination of two in-
determinately growing plum tomato cultivars (Sir Elyan and Akyra) and
three biostimulant applications (control or two concentrations of plant-
derived protein hydrolysate 2.5 or 5.0 ml L−1). Treatments were ar-
ranged in a randomized complete-block design with three replicates per
treatment amounting to a total of 18 experimental unit plots. Each
experimental unit consisted of a 10 m2 plot area containing 30 plants
(n = 540 plants).
The commercial plant-derived pH biostimulant Trainer® (Italpollina
S.p.a, Rivoli Veronese, Italy) obtained through enzymatic hydrolysis of
proteins from legume seeds was used in the current experiment.
Trainer® contains mostly soluble peptides, carbohydrates (90 g kg−1)
and free amino acids (Colla et al., 2015; Lucini et al., 2015).
The treated plants were uniformly sprayed nine times during the
growing cycle at 10-day intervals using a 16-L stainless steel sprayer
‘Vibi Sprayer’ (Volpi, Piadena, Italy). Foliar application was initiated
15 days after transplanting (17 August). The two Trainer® concentra-
tions were used on the basis of the manufacturer’s recommendations.
Crop was pruned at the 6th truss stage. Pollination was facilitated by
bumble bees introduced by means of a ‘Natupol’ hive (Koppert Italia
S.R.L., Bussolengo, Italy) upon full flowering of the first truss. During
the growing season, pests (spider mites, leaf miners, aphids) and pa-
thogens (late blight, downy mildew, grey mould) were controlled based
on standard commercial practices used in Italy.
2.3. Nutrient solution management
Fertilizer was applied through the drip irrigation system consisting
of drip lines placed 5 cm away from the tomato plants, with in-line
emitters located 0.30 m apart and an emitter flow of 2.4 l h−1. The
nutrient solution was delivered through fertigation with a composition
(in mmol L−1) of 11.0 N-NO3−, 1.5 S, 1.0 P, 6.0 K, 4.0 Ca, 2.0 Mg, 1.5
N-NH4+ and (in μmol L−1) 20.0 Fe, 9.0 Mn, 0.3 Cu, 1.6 Zn, 20 B and
0.3 Mo with an electrical conductivity of 1.8 dS m−1 and a pH of 6.2.
Fertigation was performed once per day. When more than one irrigation
event per day was necessary, plants were fertigated in the morning
followed by irrigation event(s) with plain water until the end of the day.
This strategy was used to avoid a buildup of salts in the rhizosphere.
2.4. Plant growth measurement, yield and yield components
At day 57 after transplanting (DAT; 28 September) the plant height
and number of leaves per plant were recorded on 15 plants per ex-
perimental unit. Furthermore, fully ripe fruits were harvested at 72
DAT (13 October) and continued until the end of the experiment.
During the harvest period, the number of fruits, mean weight and
marketable yield were recorded on twenty plants located at the central
part of each experimental unit. The marketable yield was expressed in
kg m−2. At the end of the experiment (14 November, 104 DAT) 15
plants per plot were separated into stems and leaves and their tissues
were dried at 80 °C for 72 h until they reached a constant weight to
determine the corresponding dry biomasses. Dry biomass was equal to
the sum of leaves and stems and was expressed in g m−2.
Y. Rouphael et al.
2.5. Collection of samples and fruit quality assessment
The fruits of the third truss were analyzed for quality parameters.
Equatorial and meridian diameters (mm) were determined at harvest
and used to calculate the fruit shape index. Tomato fruits were excised
and homogenized in a Waring® blender (2 l capacity; Model HGB140,
CA, USA) for 1 min under low speed to avoid foaming. Part of the
homogenate was filtered through double cheesecloth and the total so-
luble solids (TSS) content and also juice pH of the filtered liquid were
measured on a digital Atago N1 refractometer (Atago Co. Ltd., Tokyo,
Japan) and a pH meter (HI-9023; Hanna Instruments, Padova, Italy),
respectively. The rest of the fresh homogenate samples were transferred
to 50 ml falcon tubes, instantly dip-frozen in liquid nitrogen, and then
stored at −80 °C for further chemical analyses. The fruits dry matter
percentage was also determined in triplicates as a percentage of fresh
mass following fruit desiccation to constant weight in a forced-air oven
at 80 °C for 72 h, and weighed using an analytical balance (Denver
Instruments, Denver, Colorado, USA).
Two hundred milligrams of freeze dried fruits were extracted with
distilled water and the hydrophilic fraction (HAA) was measured with
the N,N-dimethyl-p-phenylenediamine (DMPD) method (Fogliano et al.,
1999). The lipophilic fraction (LAA) was also extracted from freeze-
dried fruits (200 mg) with methanol, and antioxidant activity of this
extract was measured with the 2,2ʹ-azinobis 3-ethylbenzothiazoline-6-
sulfonic acid ABTS method (Pellegrini et al., 1999). The absorbance of
the HAA and LAA solutions was measured at 505 and 734 nm, re-
spectively. HAA and LAA were expressed as mmol ascorbic acid and as
mmol of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid) per 100 g of dry weight, respectively.
Lycopene content was determined as described by Sadler et al.
(1990). Briefly, 5 g of homogenized sample was extracted adding 50 ml
of a mixture of hexane/acetone/ethanol (2:1:1, v/v/v) for 30 min. The
total lycopene content expressed in mg 100 g−1 fresh weight was ob-
tained by measuring the absorbance of the lycopene hexane fraction at
472 nm. Pure lycopene (Sigma, St. Louis, MO) was used for the pre-
paration of calibration curves.
The total phenolic content in methanolic extracts was determined
using the Folin-Ciocalteau procedure (Singleton et al., 1999) with gallic
acid as a standard. A 100 μl aliquot of the supernatant was combined
with 500 μl of Folin-Ciocalteau’s reagent (Sigma Aldrich Inc, St Louis,
MO, USA) and 400 μl of 7.5% sodium carbonate/water (w/v). Ab-
sorption was measured after 30 min at 765 nm using a UV–vis spec-
trophotometer, and the result was expressed as mg gallic acid (Sigma
Aldrich Inc, St Louis, MO, USA) per 100 g dry weight.
The total ascorbic acid defined as ascorbic acid and dehy-
droascorbate acid was also assessed by spectrophotometric detection.
The assay is based on the reduction of Fe3+ to Fe2+ by ASA and the
spectrophotometric detection of Fe2+ complexes with 2,2-dipyridyl
(Kampfenkel et al., 1995). The absorbance of the solution was mea-
sured at 525 nm, and the results were expressed as mg ascorbic acid on
100 g fresh weight.
2.6. Net photosynthesis measurement
At 90 DAT (31 October), the leaf net CO2 assimilation rate (ACO2;
μmol CO2 m−2 s−1) was measured with a portable gas exchange ana-
lyzer (LCA-4; ADC BioScientific Ltd., UK) equipped with a broadleaf
chamber (cuvette window area of 6.25 cm2). This physiological mea-
surement was carried out within 2 h across solar noon (i.e. between
11.00 and 13.00) on the youngest fully expanded leaves, using six re-
plicates for each treatment. Photosynthetically active radiation, relative
humidity and carbon dioxide concentrations were set at ambient values
(600 ± 30 μmol photons m−2 s−1, 50 ± 5% and 366 ± 8 ppm, re-
spectively) and the flow rate of air was 400 ml s−1.
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Scientia Horticulturae 226 (2017) 353–360
2.7. Mineral content analysis
The total nitrogen concentration of the leaf samples was determined
by the Kjeldahl method (Bremner, 1965), following mineralization with
sulphuric acid (96%, Carlo Erba Reagents, Milan, Italy) in the presence
of potassium sulfate and low concentration of copper catalyst. For the P,
K, Ca, Mg and Na analysis, 250 mg of finely ground dried plant tissues
(leaf and fruit) were suspended in 50 ml of ultrapure water (Milli-Q,
Merck Millipore, Darmstadt, Germany) and subjected to four freeze-
thaw cycles in liquid nitrogen followed by shaking water bath (Sha-
keTemp SW22, Julabo, Seelbach, Germany) at 80 °C for 10 min. The
mixture was centrifuged at 6000 rpm for 10 min (R-10 M, Remi Elek-
trotechnik Limited, India), then filtered through a 0.20 μm filter paper
(Whatman International Ltd., Maidstone, U.K.), as described previously
by Rouphael et al. (2017b). Potassium, Ca, Mg and Na were separated
by ion chromatography (ICS-3000, Dionex, Sunnyvale, CA, USA) and
quantified through an electrical conductivity detector. Chromato-
graphic separation was achieved in isocratic mode on an IonPac CS12A
analytical column (4 × 250 mm, Dionex, Corporation) equipped with
an IonPac CG12A precolumn (4 × 250 mm, Dionex, Corporation) and a
self-regenerating suppressor CERS500 (4 mm, Dionex, Corporation).
The Phosphorus content was also measured through ion chromato-
graphy coupled to a conductivity detector. A IonPac ATC-HC anion trap
(9 × 75 mm), and a AS11-HC analytical column (4 × 250 mm)
equipped with an AG11-HC precolumn (4 × 50 mm) and a self-re-
generating suppressor AERS500 (4 mm) were used for separation.
2.8. Statistical analysis of data
Experimental data were subjected to analysis of variance (two way-
ANOVA) using the software package SPSS 10 for Windows 2001. To
separate treatment means within each measured parameter, the
Duncan’s Multiple Range Test was performed at P ≤ 0.05. Principal
component analysis (PCA) was performed on fruit morphometric
components, mineral profile and juice phytochemical parameters to
depict relationships among treatments and variables and also to in-
dividuate quality traits that were most effective in discriminating be-
tween cultivars and biostimulant application. PCA is a multivariate
statistical method aimed to summarize and extract trends when several
variables are used, by formulating new variables (i.e. Principal
Components; PCs) correlated to the original ones (Lawless and
Heymann, 2010). The PCs with eigenvalues greater than 1 were se-
lected (Dunteman, 1989), and loadings greater than |0.6| indicated
significant correlations between the original variables and the extracted
components (Matus et al., 1996). The PCA outputs include variable
loading to each selected component and treatment component scores.
Analyses and calculations were carried out using the appropriate
functions within SPSS 10 (SPSS Inc., USA).
3. Results and discussion
3.1. Growth and yield responses
Plant height and leaf number before the beginning of fruit harvest as
well as shoot biomass and marketable yield were influenced by cultivar
and biostimulant treatments with no significant cultivar × biostimu-
lant interaction, whereas the mean fruit mass and fruit number incurred
significant cultivar × biostimulant interaction (Table 1). Our data in-
dicated that PH biostimulant application at 5.0 ml L−1 enhanced plant
height and leaf number by 14.3% and 21.8%, respectively, compared to
PH applications at 2.5 ml L−1 and untreated plants. Shoot biomass
demonstrated a tendency for increase in response to PH biostimulant
application rate; however, increase was significant only at 5.0 ml L−1,
at which rate shoot biomass was higher by 19.6% over untreated plants,
while no significant difference was found between the 2.5 and
5.0 ml L−1 rates (Table 1). Similarly to the effects on plant growth
Y. Rouphael et al. Scientia Horticulturae 226 (2017) 353–360

Table 1
Analysis of variance and mean comparisons for plant height and leaf number before the beginning of fruit harvest (57 days after transplanting), and total dry biomass of shoots,
marketable yield and yield components of two tomato cultivars treated with protein hydrolysate-based biostimulant at three rates of foliar application.

Source of variance Plant height (m) Leaf number (n. plant−1) Shoot biomass (g d w m−2) Marketable yield (kg m−2) Marketable fruit

Mean weight (g fruit−1) Number (no. m−2)

Cultivar *** *** *** ** *** ***

Biostimulant *** ** * ** *** ns
Cultivar × Biostimulant ns ns ns ns *** *

Cultivar
Akyra 2.5 a 58.1 a 438.9 a 10.1 b 31.7 b 319.5 a
Sir Elyan 2.0 b 43.4 b 317.4 b 11.4 a 84.6 a 134.3 b

Biostimulant (ml L−1)

0.0 2.1 b 46.7 b 341.0 b 9.8 b 52.6 c 217.2
2.5 2.1 b 47.9 b 385.6 ab 10.6 b 58.0 b 228.6
5.0 2.4 a 57.6 a 407.8 a 11.9 a 63.8 a 234.9

Cultivar × Biostimulant
Akyra 0.0 ml L−1 2.4 54.5 391.5 9.1 31.1 d 293.4 b
Akyra 2.5 ml L−1 2.4 56.0 450.3 10.5 31.6 d 330.8 a
Akyra 5.0 ml L−1 2.6 63.8 474.9 10.8 32.3 d 334.2 a
Sir Elyan 0.0 ml L−1 1.9 38.9 290.7 10.5 74.1 c 141.0 c
Sir Elyan 2.5 ml L−1 1.8 39.9 321.0 10.7 84.4 b 126.4 c
Sir Elyan 5.0 ml L−1 2.1 51.3 340.8 12.9 95.4 a 135.6 c

a
ns,*,**, *** Nonsignificant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple-
range test (P = 0.05).

parameters, the mean marketable yield in PH-treated plants at confirmed by several authors (Colla et al., 2014; Matsumiya and Kubo,
5.0 ml L−1 was significantly higher than that obtained from tomato 2011; Ugolini et al., 2015) who demonstrated that leaf/root applica-
plants treated at 2.5 ml L−1 or untreated plants, with respective in- tions of plant-derived PHs containing amino acids, carbohydrates and
creases by 12.3% and 21.4% (Table 1). Interestingly, the higher mar- ‘root hair promoting peptides’ exhibited auxin- and in some cases gib-
ketable production observed on plants treated with the legume-derived berellin-like activity, thus promoting plant growth, stimulating flow-
PH biostimulant at 5.0 ml L−1 and to a lesser extent at 2.5 ml L−1, in ering, fruit setting and growth leading to a better yield.
comparison to the untreated plants, was due to an increase in the
number of fruits per plant in the small fruit size cultivar Akyra and to a
3.2. Net CO2 assimilation rate and leaf nutrient composition
higher fruit weight in the medium fruit size cultivar Sir Elyan (Table 1).
Furthermore, irrespective of the biostimulant treatment our results
A significant effect of legume-derived PH application was observed
showed that Akyra was characterized by a more vigorous vegetative
on net leaf CO2 assimilation rate (ACO2; Fig. 1). ACO2 was higher by
growth (i.e. higher plant height, leaf number and shoot biomass) in
20.7% in the biostimulant treatment under both concentrations (avg.
comparison to Sir Elyan, whereas the latter cultivar demonstrated
11.7 μmol CO2 m−2 s−1) compared to untreated tomato plants
higher marketable yield (Table 1).
(9.7 μmol CO2 m−2 s−1; Fig. 1).
The increase in yield and growth responses of tomato plants has
Neither cultivar nor biostimulant treatment had a significant effect
been reported in limited research studies testing the action of PHs under
on total N, S and Ca concentration in leaves (avg. 30.7, 7.7 and
greenhouse conditions (Colla et al., 2015). These biostimulant effects
27.8 g kg−1 dw, respectively; Table 2). Irrespective of biostimulant
on growth and crop productivity appear to be distinct from the nutri-
treatment, K and Na concentrations in leaf tissue were more pro-
tional effect of fertilizer (i.e., N; Calvo et al., 2014). Our results on the
nounced in Akyra compared to Sir Elyan (Table 2). The PH biostimulant
foliar application of plant-derived pH at 5.0 ml L−1 were consistent
treatment at 5.0 ml L−1, averaged over cultivars, affected the P and Mg
with the findings of Colla et al. (2014) who demonstrated that in-
creasing the concentration of commercial legume-derived pH Trai-
ner®elicited significant increase in four-week-old tomato shoot, root and
total biomass amounting to 19.5%, 27.5%, and 20.5%, respectively.
The typical short-time stimulation effect previously observed in PH-
treated tomato plants (Parrado et al., 2008; Colla et al., 2014) was
confirmed in our long period trial when the plants reached maturity. In
fact, foliar application of legume-derived PH at 5.0 ml L−1 increased
marketable yield of Akyra and Sir Elyan by modulating yield compo-
nents differently depending on cultivar (higher number of fruits in
Akyra as opposed to increase of fruit mean weight in Sir Elyan).
A putative mechanism behind the stimulation of crop performance
observed in response to foliar applications of legume-derived PH at
5.0 ml L−1 could involve the augmented presence of signaling mole-
cules, such as soluble peptides, oligopeptides and free amino acids,
which are typical components of Trainer® (Colla et al., 2015). The low-
molecular size peptides as well as free amino acids are easily absorbed
by both leaves and roots and can act as signaling compounds in the Fig. 1. Mean effects of cultivar and biostimulant application rate on net CO2 assimilation
rate of tomato leaves. Different letters indicate significant differences according to
regulation of plant growth and development, promoting endogenous
Duncan’s test (P < 0.05). The values are means of three replicates. Vertical bars
phytohormonal biosynthesis (Cavani and Ciavatta, 2007). This was also indicate ± SE of means.

356
Y. Rouphael et al. Scientia Horticulturae 226 (2017) 353–360

Table 2 and morphology (i.e. architecture) of the root system, facilitating nu-
Analysis of variance and mean comparisons for macronutrients and sodium content in trient uptake as a consequence of the increased root absorbing area
leaves of two tomato cultivars treated with protein hydrolysate-based biostimulant at
(Ertani et al., 2013; Rouphael et al., 2017b). A possible mechanism
three rates of foliar application.
involved in the increase of root hair numbers and length could be re-
Source of variance Mineral composition (g kg−1 dw) lated to the stimulation of auxin production by the soluble peptides
present in the plant-derived PH Trainer®. Moreover, in a recent study,
N P S K Ca Mg Na Ertani et al. (2017) demonstrated how an alfalfa-based PH tested on
Cultivar ns ns ns *** ns ns ** tomato plants at two different concentrations (0.1 and 1 ml L−1) in-
Biostimulant ns * ns * ns ** ns duced higher expression of nutrient transporters (NTP2, SULTR2, PT2),
Cultivar × Biostimulant ns ns ns ns ns ns ns and elicited higher foliar accumulation of mineral elements, in parti-
Cultivar cular P and K. The biostimulant-mediated joint effects of increased root
Akyra 31.2 2.6 7.8 40.6 a 27.5 7.9 3.5 a surface area and enhanced nutrient transporter activity may explain the
Sir Elyan 30.3 2.6 7.6 29.2 b 28.1 8.2 2.3 b improved uptake of P, K, and Mg found in the leaves of tomato plants
Biostimulant (ml L−1) treated with plant-derived PH Trainer®.
0.0 29.1 2.2 b 6.8 32.8 b 25.6 6.8 b 3.1
2.5 30.7 2.5 b 8.2 34.4 ab 28.2 7.8 b 2.8
5.0 32.5 3.0 a 8.2 37.5 a 29.7 9.7 a 2.8 3.3. Fruit quality characteristics
a
ns,*,**, *** Nonsignificant or significant at P ≤ 0.05, 0.01, and 0.001, respectively.
Different letters within each column indicate significant differences according to Whereas an overall increase in tomato yield was observed in bios-
Duncan’s multiple-range test (P = 0.05). timulant-treated plants, improving fruit quality by foliar applications of
legume-derived PH is a challenging and pending issue as increased
concentrations in leaf tissue, which were higher by 27.6% and 32.9% demand for horticultural commodities of premium flavor quality has
on average than respective concentrations in PH 2.5 ml L−1 and control characterized consumer behavior in recent years. In the current study,
treatments. Moreover, the highest K concentration was also observed in both cultivar and biostimulant application significantly affected the
response to the 5.0 ml L−1 treatment, though the difference with the total soluble solids (TSS) content, whereas the fruit shape index (SI),
2.5 ml L−1 treatment was non-significant (Table 2). dry matter content (DM) and juice pH were affected by cultivar only
It is well established that the amino acid and peptide components (Table 3).
typical of Trainer® improve the plant’s photosynthetic activity and The Sir Elyan cultivar showed a lower fruit SI, dry matter content
subsequently lead to better yields (Colla et al., 2015). The improved and juice pH in comparison to the Akyra cultivar (Table 3). The lack of
functioning of the photosynthetic apparatus with both concentrations changes between treated and untreated plants on fruit SI could be ex-
of the PH biostimulant could be associated to a better uptake and pected since it constitutes an inherent quality trait linked to tomato
translocation of Mg. In fact, Mg is an essential macronutrient for plant fruit morphology, governed predominantly by genetic material (geno-
growth and development and is involved in a wide range of biochemical type), and little affected by environmental or cultural practices (i.e.
and physiological activities, including pigment synthesis, energy me- biostimulant application) (Kyriacou et al., 2017). Concerning sugar
tabolism and photosynthetic carbon fixation, while it forms the core content, which is a key quality attribute for consumer satisfaction
component of chlorophylls’ porphyrin ring (Gransee and Führs, 2013; (Kader, 2002), our results showed that highest juice TSS content was
Hermans et al., 2011; Kumar et al., 2015). obtained in PH -treated plants at either concentration (Table 3). These
In addition to improved photosynthesis, legume-derived PH espe- findings are consistent with previous work in which treatment with two
cially at 5.0 ml L−1 influenced the capacity of tomato plants to absorb, natural biostimulants: one derived from enzymatic hydrolysis of alfalfa
translocate and accumulate macronutrients in leaf tissue. In the current plants (applied at 25 and 50 ml L−1) and the other obtained by cool
experiment, tomato plants treated with PH were able to maintain a extraction of red grapes (applied at 50 and 100 ml L−1), elicited an
better nutritional status, evidenced as higher P, K and Mg concentration increase in the content of carbohydrates such as glucose and fructose of
in response to the 5.0 ml L−1 application rate. The enhanced nutritional greenhouse chili pepper (Capsicum chinensis L.; Ertani et al., 2014).
status has often been associated with the ability of plant-derived PH It is well known that minerals command key roles in the human
(e.g. legume-seeds hydrolysate or degraded soybean meal products; body’s metabolism, thus human nutrition deficient in these bioactive
Colla et al., 2014; Matsumiya and Kubo, 2011) to modify the growth constituents can result in nutritional disorders and expression of disease
symptoms (Gharibzahedi and Jafari, 2017). Levander (1990) reported

Table 3
Analysis of variance and mean comparisons for shape index, dry matter, total soluble solids (TSS), juice pH and mineral composition in fruits of two tomato cultivars treated with protein
hydrolysate-based biostimulant at three rates of foliar application.

Source of variance Shape index Dry matter (%) TSS (°Brix) pH Mineral composition (g kg−1 dw)

P K Ca Mg Na

Cultivar *** ** *** * * ** *** *** *

Biostimulant ns ns * ns ns * ns ** ns
Cultivar × Biostimulant ns ns ns ns ns ns ns ns ns

Cultivar
Akyra 0.75 a 6.84 a 4.90 a 4.48 a 4.65 b 35.15 a 7.30 a 1.34 a 0.86 a
Sir Elyan 0.68 b 6.16 b 4.02 b 4.40 b 6.68 a 31.78 b 4.54 b 0.92 b 0.66 b

Biostimulant (ml L−1)

0.0 0.72 6.46 4.22 b 4.42 6.18 32.28 b 5.59 0.99 b 0.87
2.5 0.71 6.50 4.50 a 4.43 5.47 32.44 b 5.97 1.14 ab 0.73
5.0 0.72 6.55 4.67 a 4.46 5.34 35.67 a 6.19 1.27 a 0.68

a
ns,*,**, *** Nonsignificant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple-
range test (P = 0.05).

357
Y. Rouphael et al.
that fruits and vegetables contribute normally by 11%, 35%, 7% and
24% to the human dietary intake of total P, K, Ca and Mg, respectively.
Taking into account the high annual per capita consumption of tomato,
ranging from 31 kg in Europe (with the exception of Italy; 60 kg) to
42 kg in North America (FAOSTAT, 2014), the contribution of tomato
fruit to human dietary intake of vitamins and especially minerals is
highly significant. Among the macroelements studied, potassium was
by far the most abundant mineral constituent in the fruit of both tomato
cultivars, followed by calcium, phosphorus, magnesium and finally
sodium (Table 3). When averaged over biostimulant application, sig-
nificant variation between the two tomato cultivars was recorded for all
macronutrient and sodium, with the highest concentrations of K, Ca,
Mg and Na recorded in Akyra, whereas the opposite was observed for P
(Table 3). Concerning the influence of legume-derived PH application
on fruit mineral profile, significant variation was recorded for K and
Mg, while no significant differences among biostimulant applications
were observed for P, Ca and Na (Table 3). Foliar application of legume-
derived PH increased K and Mg significantly at the 5.0 ml L−1 appli-
cation rate, thereby enhancing the nutritional value of fresh-tomato
fruits. Our current findings indicated that the increase in tomato fruit
concentration (K and Mg) induced by the application of legume-derived
PH at 5.0 ml L−1 could be mediated through multiple mechanisms in-
volving: (1) the presence of bioactive compounds in legume-derived PH
Trainer® (soluble peptides, carbohydrates and free amino acids) which
may have increased sink strength and influenced the movement of
nutrient substrates, including minerals, within the plant (Calvo et al.,
2014), (2) the greater uptake of minerals through a stimulation of root
growth (Billard et al., 2014; Colla et al., 2014), and also (3) the higher
expression of nutrient transporters in cell membranes (Ertani et al.,
2017).
Antioxidant activities and biologically active compounds were also
influenced by cultivar and biostimulant treatment with no significant
cultivar × biostimulant application interaction (Table 4). The anti-
oxidant activities, lycopene, total phenols and ascorbic acid contents
recorded in the present study were cultivar-dependent with the highest
values observed in the mini plum-tomato Akyra (Table 4). The strong
influence of genetic factors on antioxidant capacity and bioactive
compounds has been previously demonstrated in greenhouse tomatoes
(Fanasca et al., 2006a,b). Antioxidant potential is a crucial parameter of
the functional quality of vegetables and is usually linked to the syner-
getic effect of multiple low-molecular weight antioxidants (e.g., vitamin
C and phenolic compounds) (Colonna et al., 2016). In the current ex-
periment, the LAA and HAA of fresh tomato ranged from 0.28 to
1.23 mmol Trolox eq. 100 g−1 dw and from 1.04 to 2.54 mmol ascorbic
acid eq. 100 g−1 dw, with the highest levels of LAA and HAA, averaged
Table 4
Analysis of variance and mean comparisons for lipophilic and hydrophilic antioxidant activities, lycopene, total phenols and total ascorbic acid in fruits of two tomato cultivars treated
with protein hydrolysate-based biostimulant at three rates of foliar application.
Source of variance Antioxidant capacity
Lipophilic (mmol Trolox eq. Hydrophilic (mmol ascorbate
100 g−1 dw) eq. 100 g−1 dw)
Cultivar * **
Biostimulant *** * ***
Cultivar × Biostimulant ns ns
Cultivar
Akyra 0.85 a 2.10 a
Sir Elyan 0.60 b 1.34 b
Biostimulant (ml L−1)
0.0 0.30 c 1.34 b
2.5 0.78 b 1.63 b
5.0 1.08 a 2.17 a
a
ns,*,**, *** Nonsignificant or significant at P ≤ 0.05, 0.01, and 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple-
range test (P = 0.05).
358
Scientia Horticulturae 226 (2017) 353–360
over cultivars, recorded in response to plant-derived PH applications at
5.0 ml L−1. Contrary to total phenols content, the total ascorbic acid
and lycopene contents were significantly influenced by biostimulant
treatments. Compared to the fruit of untreated plants, lycopene content
was increased by 34.9% and 18.0% in response to foliar application of
commercial legume-derived PH delivered at 5.0 and 2.5 ml L−1, re-
spectively. Similarly, the total ascorbic acid in PH-treated plants at
5 ml L−1 was significantly higher by 27.3% than that recorded in
2.5 ml L−1 treated and untreated tomato plants (Table 4). These find-
ings are in agreement with those of Ertani et al. (2014), who reported
that ascorbic acid increased 2.5-fold in green chili pepper fruits treated
with alfalfa-derived biostimulant at 25 ml L−1, whilst it increased 1.28-
fold in red chili pepper fruits following the application of red grape-
derived biostimulant at 100 ml L−1, in comparison to untreated plants.
The synthesis and accumulation of antioxidant compounds such as
ascorbic and lycopene could be attributed to direct or indirect effects of
biostimulant applications on the biosynthesis and turnover of anti-
oxidants in plant tissues. Plant-derived PH applications may have pro-
moted the activity of specific enzymes (direct effects) involved in an-
tioxidant homeostasis in cells as reported by Ertani et al. (2014).
Moreover, indirect effects such as the changes of K uptake and leaf
coverage may have influenced the accumulation of antioxidants in
fruits. Lycopene content was significantly correlated (Pearson’s coeffi-
cient = 0.915; p < 0.05) to K concentration in the fruits (data not
shown). The same type of correlation between lycopene and K con-
centration has been previously reported in tomatoes (Fanasca et al.,
2006a,b; Taber et al., 2008). Potassium is essential for the pyruvic ki-
nase and acetic thiokinase enzymes that are directly involved in lyco-
pene synthesis. Potassium may also affect lycopene synthesis as a co-
factor facilitating enhanced enzymatic activity in carbohydrate meta-
bolism, thus providing necessary substrates for terpenoid synthesis in
tomato as terpenes via the isoprenoid pathway can be formed from
either carbohydrates or lipids (Fanasca et al., 2006a; Taber et al., 2008;
Tomás-Barberán and Robins, 1997). Similarly, ascorbic acid increased
with increasing levels of K in several crops (Lester, 2006; Rouphael
et al., 2012). Moreover, climate factors have an important role in an-
tioxidant accumulation in tomato fruits. It is well known that high fruit
temperatures can degrade lycopene and impede lycopene synthesis
(Taber et al., 2008); similarly, ascorbic acid declined with increasing
temperature in tomato fruits (Lester, 2006). The biostimulant-mediated
increase in leaf number may have enhanced fruit shading thereby
moderating fruit temperature and enhancing antioxidant compounds.
The incurred accumulation of these health-promoting metabolites, ly-
copene in particular, in response to legume-derived PH-applications
could be considered an added value to tomato’s contribution toward
Lycopene (mg 100 g−1 fw) Total phenols (mg gallic acid Total ascorbic acid
eq. 100 g−1 dw) (mg 100 g−1 fw)
*** *** ***
ns ***
ns ns ns
69.2 a 27.0 a 109.1 a
40.7 b 18.3 b 69.9 b
46.7 c 22.5 76.9 b
55.1 b 22.7 87.1 b
63.0 a 22.9 104.4 a
Y. Rouphael et al. Scientia Horticulturae 226 (2017) 353–360

Table 5
Eigen values, relative and cumulative percentage of total variance, and correlation
coefficients for each tomato trait with respect to the two principal components (PC1 and
PC2).

Principal components PC1 PC2

Eigen value 12.6 2.1

Relative variance (%) 79.3 13.7
Cumulative variance (%) 79.3 93.0

Eigen vectors
Lycopene 0.988 0.070
Total soluble solids 0.988 0.088
Mg 0.984 0.137
Total ascorbic acid 0.977 0.165
Ca 0.976 −0.182
Dry matter 0.964 −0.166
Fruit number 0.960 −0.161
Total phenols 0.940 −0.164
Juice pH 0.928 0.206
K 0.884 0.268
Hydrophilic antioxidant activity 0.883 0.467
P −0.873 0.044
Fruit weight −0.864 0.375
Shape index 0.781 −0.441
Na 0.491 −0.841
Lipophylic antioxidant activity 0.591 0.802

Boldface factor loadings indicate the most relevant characters for each principal com-
ponent.

human health and longevity (Erba et al., 2013). In fact, several pre-
clinical and clinical investigations have revealed that this natural pig-
ment reduces the risk or progression of diseases associated with oxi-
dative stress (Berman et al., 2015).
3.4. Principal component analysis
A comprehensive framework of biostimulant application and cul-
tivar effects on morphometric and physicochemical quality character-
istics of fresh tomato was obtained through Principal Component
Analysis (PCA). The first two PCs were associated with Eigen values
higher than one and explained cumulatively 93% of the total variance,
with PC1 and PC2 accounting for 79.3% and 13.7%, respectively
(Table 5). PC1 was highly and positively correlated with HAA, bioactive
compounds (lycopene, total ascorbic acid and phenols), DM, TSS, K, Ca,
Mg, juice pH and fruit SI; it was negatively correlated with fruit weight
and P (Table 5). PC2 was positively correlated with LAA and negatively
associated with Na content (Table 5). Moreover, the loading plot in
Fig. 2A illustrates the correlation among variables, where two vectors
with an angle lower than 90° are positively correlated and two vectors
with an angle higher than 90° are negatively correlated. For instance,
variation in TSS content was most closely aligned with lycopene, and
variation in ascorbic acid was more strongly correlated to Mg content
(narrower angle between the corresponding vectors) rather than LAA.
The correlation between sugars (TSS) and total ascorbic acid could be
expected since soluble hexoses are known precursors of ascorbate
synthesis (Nagy, 1980).
The effectiveness of PCA plotting in genetic characterization and in
preharvest and postharvest quality experiments has been documented
in several scientific papers (Kyriacou et al., 2016; Rouphael et al., 2016,
2017c). This was also the case in the present study, since PCA scores in
Fig. 2B have introduced concerted information on the tomato quality
traits in relation to cultivars and biostimulant application. The PC1 and
PC2 score plot separates and categorizes biostimulant treatments and
cultivars into four groups (quadrants). The upper left quadrant of the
negative side of PC1 (Q1) included cultivar Sir Elyan treated with plant-
derived PH at 5.0 ml L−1 that delivered fruit of high weight and high P
content (Fig. 2B). A second group clustered on the positive side of PC1
(Q2) included Akyra treated with PH at both 2.5 and 5.0 ml L−1,
Fig. 2. (A) Principal component loading plot and (B) scores of principal component
analysis of quality traits of greenhouse tomato as a function of biostimulant application
rate (control = 0.0 ml L−1; 1 = 2.5 ml L−1; 2 = 5.0 ml L−1), and cultivars (Akyra and
Sir Elyan). LAA, lipophylic antioxidant activity; HAA, hydrophilic antioxidant activity;
AA, total ascorbic acid; TSS, total soluble solids; DM, dry matter; SI, shape index.
representing tomato fruits with premium quality (high LAA, lycopene,
K and Mg contents). The lower left quadrant (Q3) depicted the treat-
ments (Sir Elyan untreated or treated at 2.5 ml L−1) of lowest quality
traits. Finally, treatments in Q4 had the highest Na and fruit SI en-
compassing the untreated Akyra (Fig. 2B). Moreover, score plot clearly
separates the two cultivars along PC1 with Akyra concentrating most of
the flavor and functional quality traits while Sir Elyan stands out for its
fruit weight and P content. The biostimulant applications are clustered
mainly in respect to PC2, with the 5.0 ml L−1 application on the posi-
tive side of PC2 that is characterized by improved size, flavor or
functional quality attributes. Overall, the PCA outputs may provide the
basis for a more in-depth approach to elucidate the influence of culti-
vars and biostimulant treatments on quality characteristics of green-
house tomato.
4. Conclusions
The increasing pressure on vegetable farmers to intensify agri-
cultural production while maintaining premium quality represents a
strong stimulus for scientists to propose alternative sustainable tech-
nologies for the improvement of vegetable quality, in order to meet
consumer demand. Greenhouse tomato crop performance and the
configuration of fruit quality was analyzed in a multifactorial approach
accounting for the effects of biostimulant treatments and cultivars.
Foliar applications of legume-derived PH Trainer® at 5.0 ml L−1 in-
creased marketable yield of Akyra and Sir Elyan by modulating yield
components (number or fruits and mean weight) differently depending
on cultivars. Application of the biostimulant, especially at the high
dose, was capable of maintaining higher photosynthesis and a better
nutritional status for the plants. Our results also demonstrated, that

359
Y. Rouphael et al.
differences in total soluble solids content and functional fruit quality
traits were identified between control and plant-derived PH treated
tomato plants at 2.5 and 5.0 ml L−1. Key quality attributes of green-
house fresh tomato (TSS, LAA, HAA, total ascorbic acid lycopene, K and
Mg contents) were improved by the applications of legume-derived PH
Trainer®.
Acknowledgement
The authors are grateful to Dr. Alfonso Marilongo for his assistance
in the greenhouse experiment and also to Dr. Antonio Pannico for the
leaf gas exchange measurements.
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